Because cytomegalovirus (CMV) may contribute to restenosis and atherosclerosis, and because smooth muscle cells (SMCs) are involved in these disease processes, we examined CMV/SMC interactions. Using confocal microscopy to identify a redox sensitive fluorescent marker, we found that CMV infection of SMCs generates intracellular reactive oxygen intermediates (ROIs). CMV also activated the cellular transcription factor NF kappaB, as demonstrated by increased NFkappaB binding to DNA (electrophoretic mobility shift assay). Antioxidants inhibited activation, suggesting a role of ROIs in CMV-induced NFkappaB activation. By using antioxidants to assess the role of ROIs in modulating virally-mediated effects, we also found CMV-induced ROIs: 1) are critical to the transactivation of the viral major immediate promoter (MIEP) by its immediate early protein IE72 (determined by cotransfection of an IE72 expression vector and a reporter gene downstream of the MIEP); 2) are necessary for IE72 expression (determined by immunocytochemistry) and viral replication (determined by viral titer assay on indicator cells) following CMV infection of SMCs. Because ROIs, through activation of NFkappaB, also can induce expression of cellular genes involved in immune and inflammatory responses, the ROI response to CMV infection may also represent a parallel survival mechanism that has evolved in the host cell to protect against viral infection. We conclude that CMV induces intracellular ROI generation within minutes after infection of SMCs, and then utilizes these ROIs to facilitate its own gene expression and replication. Conversely, antioxidants inhibit CMV IE gene expression and viral replication.